Genetically encoded fluorescent coumarin amino acids

Abstract

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine into proteins produced in eubacterial host cells such as E. coli . The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

1 - 40 . (canceled) 
     
     
         41 . A protein comprising an unnatural amino acid residue, wherein the unnatural amino acid is an L-(7-hydroxycoumarin-4-yl)ethylglycine. 
     
     
         42 . The protein of  claim 41 , wherein the protein is selected from the group consisting of a therapeutic protein, a diagnostic protein, an enzyme, and a binding protein. 
     
     
         43 . The protein of  claim 42 , wherein the protein comprises two or more unnatural amino acids. 
     
     
         44 . The protein of  claim 42 , wherein the L-(7-hydroxycoumarin-4-yl)ethylglycine is at an internal position of the protein. 
     
     
         45 . The protein of  claim 41 , wherein the protein is produced by the method of:
 a) providing a translation system comprising:
 (i) L-(7-hydroxycoumarin-4-yl)ethylglycine; 
 (ii) an orthogonal aminoacyl-tRNA synthetase (O-RS); 
 (iii) an orthogonal tRNA (O-tRNA), wherein the O-RS preferentially aminoacylates the O-tRNA with the L-(7-hydroxycoumarin-4-yl)ethylglycine; and, 
 (iv) a nucleic acid encoding the protein, wherein the nucleic acid comprises at a selected position at least one selector codon that is recognized by the O-tRNA; and, 
   (b) incorporating the L-(7-hydroxycoumarin-4-yl)ethylglycine in the protein during translation of the protein in response to the selector codon, thereby producing the protein comprising L-(7-hydroxycoumarin-4-yl)ethylglycine at the selected position.   
     
     
         46 . The protein of  claim 45 , wherein the O-RS comprises the amino acid sequence set forth in SEQ ID NO: 4, or conservative variants thereof comprising at least 90% sequence identity to SEQ ID NO: 4 with the proviso that positions 32, 67, 70, and 158 of SEQ ID NO: 4 are conserved. 
     
     
         47 . A method of using a fluorescent protein probe, the method comprising:
 providing a protein probe comprising a L-(7-hydroxycoumarin-4-yl)ethylglycine at a selected position;   exposing the protein to a changed condition; and,   detecting a change fluorescence of the protein associated with the changed condition.   
     
     
         48 . The method of  claim 47 , wherein the changed condition is selected from the group consisting of: a bimolecular interaction, a conformational change in the protein, a change in the topology of the protein bound to a membrane, a pH change, and a change in urea concentration. 
     
     
         49 . The method of  claim 48 , wherein the bimolecular interaction is a fluorescence resonance energy transfer (FRET) interaction between the protein and a fluorophore or a quencher. 
     
     
         50 . The method of  claim 47 , further comprising determining site-specific protein conformational changes by selecting the position at a particular site of interest in the protein.